A large number of previous studies have revealed that sugar chain structures bonded to proteins play an important functional role in the biological activities of the proteins. The sugar chain is also called the “face of the cell”. The sugar chain expressed on cell surface is known to participate in cell-cell interaction or signaling, development or differentiation, fertilization, cancer metastasis, etc. As for modifications of sugar chains in mammals, Asn-linked, mucin-type, proteoglycan-type glycosylation and others are typically well-known. These modifications form their respective unique sugar chain structures through distinctive biosynthesis pathways. Sugars such as fucose or sialic acid are known to be added to the non-reducing ends of such sugar chain structures.
The sialic acid is a generic name for amino group- or hydroxy group-substituted compounds of neuraminic acid, which is a special nonose having amino and carboxy groups. N-acetylneuraminic acid (Neu5Ac) having an acetylated amino group at position 5 is probably the most predominant form in the nature. Various structures such as N-glycolylneuraminic acid having a glycolyl-modified amino group at position 5 or deamino-neuraminic acid KDN are also known.
Reportedly, the sialic acid-containing sugar chain is found not only in mammals including humans and mice but in vertebrates, echinoderms, and even protists or some bacteria having gram-negative pathogenicity. This sialic acid-containing sugar chain is produced via sialyltransferase. The sialyltransferase employs sialic acid added to cytidine monophosphate (CMP) as a substrate donor to transfer the sialic acid to, for example, position 3 or 6 of galactose, position 6 of N-acetylgalactosamine, or position 8 of another sialic acid via an aldehyde group present at position 2 of the sialic acid donor. For example, the enzyme transferring sialic acid to position 3 of galactose is called α-2,3-sialyltransferase; the enzyme transferring sialic acid to position 6 of galactose or N-acetylgalactosamine is called α-2,6-sialyltransferase; and the enzyme transferring sialic acid to position 8 of another sialic acid is called α-2,8-polysialyltransferase. Of these enzymes, the α-2,6-sialyltransferase is known as enzymes ST6Gal-I and ST6Gal-II transferring sialic acid to position 6 of galactose and enzymes ST6GalNAc-I, ST6GalNAc-II, ST6GalNAc-III, and ST6GalNAc-IV transferring sialic acid to position 6 of N-acetylgalactosamine, in humans.
ST6Gal-I recognizes a N-acetyllactosamine structure (Galβ1-4GlcNAc), which is N-acetylglucosamine having galactose linked to position 4, as a substrate acceptor and therefore modifies the non-reducing end structures of some glycolipids or N-linked sugar chains. Its specificity for the substrate acceptor has been analyzed mainly using biantennary or triantennary N-linked sugar chains. According to the report, the sialic acid tends to be transferred to lactosamine on the antenna of α1,3-linked mannose (see Non Patent Literature 1). As for the preparation of the biantennary or triantennary N-linked sugar chains, these sugar chains are difficult to efficiently produce in quantity because, for example: glycosyltransferase substrates are rarely extracted from natural products; and a large-scale preparation method for the enzyme has not yet been established.
Meanwhile, α2,6-sialic acid transfer reaction for tetraantennary N-linked sugar chains has been studied using bovine-derived ST6Gal-I. Of four N-acetyllactosamine structures in the sugar chain, the N-acetyllactosamine structure β1,2-linked to α1,3-linked mannose is most susceptible to sialic acid transfer, followed by the N-acetyllactosamine structure β1,4-linked to α1,3-linked mannose and further, either of two N-acetyllactosamine structures added to α1,6-linked mannose, though no product containing four sialic acid molecules has been found (see Non Patent Literature 2). Human ST6Gal-I and, also ST6Gal-II, have been reported to have substrate specificity (see Non Patent Literatures 3 and 4). However, no study has been made on sialylation with tetraantennary N-linked sugar chains as acceptor substrates.
According to the reports, the product inhibition of ST6Gal-I by CMP is 49% inhibition (see Non Patent Literature 5) or 71% inhibition (see Non Patent Literature 6) by 0.25 mM CMP.
Meanwhile, Photobacterium damsela JT0160 (see Non Patent Literature 7), Photobacterium leiognathi JT-SHIZ-145 (see Non Patent Literature 8), and the like have been reported as bacterium-derived α2,6-sialyltransferase. None of them, however, have been studied on sialylation with tetraantennary N-linked sugar chains as acceptor substrates.
As for α2,3-sialic acid transfer reaction for tetraantennary N-linked sugar chains, tetraantennary N-linked sugar chains containing four α2,3-linked sialic acid molecules are added to glycoproteins such as erythropoietin (EPO) (see Non Patent Literature 9). According to the report, such sialylation contributes to the stability of the glycoproteins in blood (see Non Patent Literature 10). Although these structures also occur naturally, there has been no case reporting that the tetraantennary N-linked sugar chains containing four α2,3-linked sialic acid molecules were actually prepared in large amounts. This is because: EPO or the like used as a starting material is difficult to prepare in large amounts in terms of cost; and the asialo tetraantennary N-linked sugar chains used as acceptors in enzymatic synthesis are also difficult to inexpensively prepare from other natural products. Also, the glycoprotein EPO is known to have, for example, tetraantennary N-linked sugar chains containing α2,3 and α2,6 linkages together (see Non Patent Literature 11).
It has been reported as to the linking pattern of sialic acid linked to N-type sugar chains in antibody drugs or glycoprotein drugs such as cytokines that proteins having α2,6-linked sialic acid disappear from blood faster than proteins having α2,3-linked sialic acid. For clearance from blood, glycoproteins are incorporated into cells through in vivo binding to lectin molecules and finally metabolized. Thus, the glycoproteins having α2,6-linked sialic acid can be expected to be incorporated in an organ-specific manner through binding to specific lectin molecules and also to be exploited in drug delivery. Also, glycoproteins are known to be excreted into urine in the kidney, depending on molecular sizes. Reportedly, the apparent molecular size of erythropoietin increases with increase in the number of antennas in its sugar chain, leading to slow clearance from blood. Thus, the synthesis of sugar chains having α2,3-linked and/or α2,6-linked sialic acid, particularly, tetraantennary N-type sugar chains having four molecules of α2,3-linked and/or α2,6-linked sialic acids can be expected to applicable to the production of glycoprotein drugs differing in the efficiency of uptake into an organ.
Human influenza virus recognizes, for its infection, α2,6-linked sialic acid in sugar chains expressed on cell surface, whereas bird-derived influenza virus recognizes α2,3-linked sialic acid for its infection. Many viruses, also including the influenza virus, start to infect cells by recognizing the sugar chain structures of the cells to be infected. In this regard, the binding specificity of these viruses must be examined using various sugar chains. Thus, sugar chains having α2,3- or α2,6-linked sialic acid may serve as a material for study on the binding specificity of such viruses and be applicable to, for example, the detection of the viruses.